RApid Parallel Protein EvaluatoR (RAPPER), from gene to enzyme function in one day

Abstract

Cell-free transcription-translation systems offer an effective and versatile platform to explore the impact of genetic variations on protein function. We have developed a protocol for preparing linear, mutagenic DNA templates for direct use in the PURE system, enabling the fast and semi-quantitative evaluation of amino acid variations on catalytic activity and stereo-selectivity in native and engineered variants of Old Yellow Enzyme.

Revealing Additional Stereocomplementary Pairs of Old Yellow Enzymes by Rational Transfer of Engineered Residues

“…No activity towards 1 was observed with PETNR W102I, but other variants at this hotspot position and other substrates showed increased conversion . Incorporation of W116I into circular permutated OYE1 variants lowered conversion of 3 , a saturation mutagenesis library at W116 showed no hits for 1 , and biocatalytic characterisation of all 20 variants showed that W116I is not the best engineered residue for this position . Nevertheless, our NCR data support the importance of hotspot position III itself.…”

“…In addition to the above studies, a few single‐residue saturation mutagenesis, rational design or directed evolution studies that allow extraction of engineered residues exist. Taken together, several hotspot positions have been identified, and positions I, II, and III (Figure ) in particular were found to control facial selectivity, activity and substrate scope in OYE1, OYE2.6, PETNR, KYE and YqjM …”

Asymmetric Ketone Reduction by Imine Reductases

“…[14][15][16][17] Activities between 21 and 99 %w ere observed in the reduction of 1a with alcohol dehydrogenases. [18,19] In biotransformations with the in vitro expressed R-selective IRED, approximately 5% of 2a was formed (Table 2). Ar ecent example of in vitro translation/transcription (IVTT) was shown by the group of Lutz, who prepared ac ell-free mutant library of the ene reductase OYE1.…”

“…Ar ecent example of in vitro translation/transcription (IVTT) was shown by the group of Lutz, who prepared ac ell-free mutant library of the ene reductase OYE1. [18,19] In biotransformations with the in vitro expressed R-selective IRED, approximately 5% of 2a was formed ( Table 2). We used the PUREexpress In Vitro Protein Synthesis KIT from New EnglandB iolabs.…”

Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay

“…However, there are publications 28−31 suggesting that in vitro reconstitution of such systems is not impossible. Regardless, our success with flavoprotein oxidoreductases in cell-free, similar to others, 32 indicates that a large subspace of anabolic reactions can be easily reconstituted and assayed with basic TxTl systems.…”